The long term goal of this research is to understand the molecular mechanisms underlying the regulation of the homeotic genes during development. Specifically, we will investigate the regulation of the homeotic genes by the trithorax locus (trx) in Drosophila melanogaster. Genetic and molecular evidence suggests that trx encodes a positive trans-acting regulator of many of the homeotic genes in the Bithorax Complex (BX-C) and Antennapedia Complex (ANT-C). The trx gene product is required to establish and maintain the normal level of expression of these genes in their appropriate segments. We will investigate how trx exerts its effects on the homeotic genes by molecular cloning of the trx DNA sequences and by characterizing the structure of its RNA and protein products. We will also determine the effects of trx mutations on the expression of selected homeotic gene products. The major strategy for cloning trx will be chromosomal walking. Determination of the location and physical extent of the locus will involve molecular mapping of trx mutations and rescue of trx mutations by P element-mediated transformation. The primary structure of the trx gene and its RNA and protein products will be determined by Northern analysis, S1 nuclease protection, primer extension and DNA sequencing of trx cDNA clones and corresponding genomic DNA. Temporal and spatial expression patterns of trx will be determined by in situ hybridization of trx gene probes to tissue sections and by immunofluorescent staining of embryos with anti-trx antibodies. The effects of trx mutations on homeotic gene expression will also use these methods, with the available probes for selected homeotic gene products. The results of these studies will provide the foundation for further experiments to determine whether trx protein(s) interacts directly with the homeotic genes and address how trx functions in the positive regulation of these genes to effects their proper spatial expression during development.